CELLULAR PROLIFERATION AND THE CDC7 PROTEIN KINASE

细胞生物学中的一个中心问题是， 控制细胞分裂周期的各个阶段，确保 重要细胞成分的有序复制和分离。 使用酵母遗传学和动物细胞生物学获得的结果， 生物化学最近在我们的研究中取得了重大突破， 了解管制和促进加入的机制 分裂。 相比之下，对G1/S的控制知之甚少 转换和DNA复制的起始。 酵母中 酿酒酵母，似乎相同的蛋白激酶参与了 CDC 2/CDC 28/MPF调节G1/S和G2/M转换， 激酶。 我们的研究旨在确定 通过激活酵母中G1期的Cdc 28蛋白来实现运动。 的 我们研究的总体目标是了解Cdc 7的作用 蛋白质，也是一种蛋白激酶，在进入S期。 CDC 7基因 在G1后期起作用。 我们的初步结果表明，Cdc 7 p是 参与了G1期潜在的磷酸化级联反应， DNA复制的起始 我们已经发现Cdc 28免疫 复合物可以在体外磷酸化Cdc 7 p，并且Cdc 7免疫 复合物可以在体外磷酸化复制蛋白RP-A。 如果得到证实，这些研究表明Cdc 7作为一种分子， 在对DNA的承诺中活跃的调节装置 合成和启动DNA的催化装置 合成. 我们将进一步表征体外相互作用， 并试图证明它们反映了体内的细胞周期。 首先，我们发现从细菌中纯化的Cdc 7蛋白是 像激酶一样无活性。 我们将研究 翻译后修饰，包括Cdc 28的磷酸化， 以及与其他酵母蛋白的相互作用， 激酶。 Cdc 28蛋白的激酶活性在整个过程中受到调节， 通过共价修饰和与 其他蛋白质，包括细胞周期蛋白，和Cdc 7可能经历类似的过程。 修改. 其次，我们将确定Cdc 7中的氨基酸 在体内和体外被磷酸化。 我们将测试他们的 通过定点改变Cdc 7的功能来影响Cdc 7的功能 诱变和分析它们与CDC 7突变体互补的能力。 第三，我们将尝试验证RP-A是Cdc 7 p的底物， 体内和研究其他潜在的底物。

A central problem in cell biology is how the transitions between the various phases of the cell division cycle are controlled, ensuring orderly duplication and segregation of crucial cellular components. Results obtained using yeast genetics and animal cell biology and biochemistry have recently led to significant breakthroughs in our understanding of the mechanisms that regulate and promote entry into mitosis. By comparison, less is known about control of the G1/S transition and the initiation of DNA replication. In Saccharomyces cerevisiae, it appears that the same protein kinase is involved in regulating both the G1/S and the G2/M transitions, the cdc2/CDC28/MPF kinase. Our studies have been aimed at defining the events set in motion by activation of the Cdc28 protein in G1 in yeast. The overall gaol of our studies is to understand the role of the Cdc7 protein, also a protein kinase, in entry into S phase. The CDC7 gene functions late in G1. Our preliminary results suggest that Cdc7p is involved in a potential phosphorylation cascade during G1 that leads to initiation of DNA replication. We have found the Cdc28 immune complexes can phosphorylate Cdc7p in vitro and that Cdc7 immune complexes can phosphorylate the replication protein, RP-A, in vitro. If verified, these studies suggest that Cdc7 serves as a molecular link between the regulatory apparatus active in commitment to DNA synthesis and the catalytic apparatus involved in initiating DNA synthesis. We will further characterize the in vitro interactions of these proteins and try to show they reflect the cell cycle in vivo. First, we have found that Cdc7 protein purified from bacteria is inactive as a kinase. We will investigate the role of posttranslational modifications, including phosphorylation by Cdc28, and interactions with other yeast proteins in activating the Cdc7 kinase. The kinase activity of Cdc28 protein is regulated throughout the cell cycle by both covalent modifications and association with other proteins, including the cyclins, and Cdc7 may undergo similar modifications. Second, we will determine the amino acids in Cdc7 that are phosphorylated in vivo and in vitro. We will test their effect on the functions of Cdc7 by altering them by site directed mutagenesis and analysis of their ability to complement cdc7 mutants. Third, we will try to verify that RP-A is a substrate of Cdc7p in vivo and investigate other potential substrates.